Hop Proanthocyanidins for the Fining of Beer

1 Hop proanthocyanidins for the fining of beer 2 3 R. S.T. Linforth*a, K. Westwoodb, A. Somanic, N. Dohertyc, D.J. Cookc 4 5 6 aFood Sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham. 7 LE12 5RD. UK. 8 bBarth Innovations Ltd, Hop Pocket Lane, Paddock Wood, Kent, TN12 6DQ, United Kingdom 9 cBrewing Science Department, School of Biosciences, Sutton Bonington Campus, University of 10 Nottingham. 11 LE12 5RD. UK. 12 13 14 15 *Corresponding author: [email protected] 16 17 18 1 19 Abstract 20 Fining agents are used in the clarification of beers; they help to reduce the time required to 21 sediment suspended yeast cells and ensure the clarity and colloidal stability of beer. 22 Following an adventitious observation during dry-hopping experiments, we identified a 23 fining activity associated with Saaz hops. Extracts of hop cones were subsequently shown to 24 have the capacity to flocculate yeast and result in their sedimentation. This activity has since 25 been identified in extracts of many different hop varieties and, significantly in spent hops, 26 the co-product resulting from commercial extraction of hops with either CO2 or ethanol. 27 Here we illustrate the activity of the novel finings extracted from spent hops following CO2 28 extraction of Galena hops. The sediments formed on fining were compact, relative to those 29 obtained when commercial isinglass was used to fine the same beers. The hop extracts were 30 also effective in reducing 90° haze in beers under conditions designed to mimic both cask 31 ale (12°C) and lager (4°C) type applications. 32 The compounds responsible for the fining activity appear to be large (30 to 100kDa, or 33 more) polyphenols. Analysis of the polyphenols using colourimetric tests, indicated the 34 presence of proanthocyanidins. On acidic hydrolysis these generated cyanidin, which would 35 be derived from a polymer composed of catechin and epicatechin subunits. The presence of 36 these materials in spent hops offers the possibility to develop commercial products, with 37 desirable fining properties, from an existing co-product stream. Furthermore, the finings are 38 derived from a traditional ingredient of the brewing process. 39 40 41 Key words. Fining agent, hop polyphenolics, proanthocyanidin, brewing, colloidal stability. 42 2 43 1. Introduction 44 The vast majority of beers consumed worldwide are intended to be served clear, bright and 45 free from visible haze. It is important that clarity is achieved in fresh beer and that this is 46 retained through the required shelf-life, such that beers are delivered to the consumer in 47 optimal condition. Haze can be considered as the ‘absence of clarity’ and is caused by the 48 presence of small insoluble particles, typically in the μm size range, which scatter light, 49 leading to the perception of haziness (1). There are several different sources of haze in 50 beers, ranging from sporadic negative factors such as microbial infection, through 51 precipitates of relatively insoluble salts such as calcium oxalate, to the presence of colloidal 52 materials (e.g. proteins, carbohydrate polymers) which are only sparingly soluble in the beer 53 matrix and therefore have a tendency to form insoluble aggregates of material, leading to 54 colloidal instability (2). Of particular relevance are the complexes formed between 55 polyphenols and so-called haze-sensitive proteins, which are responsible for chill-haze in 56 beer (3; 4; 5) (haze material which comes out of solution when beers are refrigerated but 57 which dissolves when returned to 20°C). Ensuring the colloidal stability of beer involves 58 control of factors across the brewing process from raw materials selection, through 59 brewhouse processing and into finished beer (6). The maturation period, post-fermentation, 60 is particularly significant in this regard. Green beers contain residual yeast cells in 61 suspension, a factor which in itself can lead to haziness of beers if steps are not taken to 62 remove them. Traditionally, the clarity of lager beers has been ensured by cold-conditioning 63 them for periods of several weeks, during which the insoluble materials settle out to form 64 ‘tank bottoms’, leaving behind bright beer. In the modern day industry it is not desirable to 65 incur the costs of chilling and storing large quantities of beer, hence rapid maturation 66 processes have been developed to ensure colloidal stability of beers over much shorter 3 67 time-frames. These usually involve the use of process-aids designed to selectively remove 68 haze materials, or their precursors. Examples would be the use of PVPP to lower the 69 polyphenolic precursors of haze, or of tannic acids or silica gels to remove portions of the 70 haze-sensitive proteins in beer (7). Physical separation processes such as centrifugation and 71 filtration are also used to remove particulates, however, a combination of approaches is 72 often required in order to ensure that the loading of solids in rough beer does not lead to 73 blinding of filters, or shortening of effective filtration run times. 74 Fining agents are used to accelerate the rate of separation of suspended particles from 75 beers and in general work by cross-linking haze particles to generate larger aggregates of 76 material which settle out more rapidly to the bottom of a vessel. Finings have traditionally 77 been used most prevalently in the production of cask ales in the UK. Since cask ales contain 78 live yeast in contact with the product and undergo secondary fermentation in the trade, it is 79 necessary to clarify such beers by the addition of fining materials, such that yeast settles out 80 efficiently on completion of the secondary fermentation, to form a compact sediment. 81 Furthermore, the use of finings to treat brewery-conditioned beers has become more 82 widespread, as part of the overall strategy of ensuring colloidal stability using shorter 83 process times (8). 84 The most widely encountered finings material in brewing is isinglass, a purified protein 85 preparation extracted with dilute acid from the swim-bladders of certain species of tropical 86 or sub-tropical fish. The active ingredient is almost pure collagen (9). Isinglass acts by cross- 87 linking suspended yeast cells, via a charge-interaction, leading to their aggregation and 88 subsequent sedimentation. Isinglass carries a net positive charge at beer pH’s, facilitating its 89 interaction with the negatively charged surface of yeast cells (8; 10). The use of isinglass is 90 well established in certain applications and regions of the world, mainly because isinglass 4 91 combines several features attractive to brewers. In addition to the flocculation of yeast 92 cells, isinglass is also active against chill-haze (8), forms sediments which are compact 93 (leading to minimal beer losses and easier run-off of beers from above the sediment), 94 improves subsequent filterability of beers and has been noted to improve beer foam (8), 95 most likely due to the removal of foam negative lipid materials. However, one aspect which 96 limits the usage of isinglass is the fact that it originates from fish swim bladders, meaning 97 that products manufactured using isinglass are not suitable for vegans and are not 98 considered kosher. At one time it was proposed that residues of isinglass in beer might pose 99 a threat to fish allergy sufferers and that products would need to be labelled accordingly 100 (11; 12); however, this requirement did not become law in the EU because it was possible to 101 prove that residual isinglass levels in treated beers were extremely low, hence did not pose 102 a threat (12). Due to the aforementioned concerns, researchers have attempted to identify 103 alternative fining materials with which to treat beers and wines. These have included 104 evaluation of avian collagen and pea protein extract (10), the use of plant pectins (13), or of 105 bovine collagen (14). To date none of these approaches have been exploited commercially, 106 probably because none of the materials individually match the performance of isinglass in 107 all of its beneficial features. Thus isinglass remains the only finings material in widespread 108 brewing usage. In spite of this, there are other aspects to the use of isinglass which might be 109 improved upon when developing novel fining agents; isinglass is not an easy material to 110 disperse and mix into water. The UK is the only region with a significant market in wet 111 isinglass products whereas the remainder of the world principally uses dry isinglass powder 112 which must first be dispersed in water to the appropriate strength, prior to dosing into the 113 process. Once these solutions have been prepared they have a limited shelf-life and need to 5 114 be stored refrigerated (4-10°C) to retain activity; at higher temperatures collagen rapidly 115 denatures to inactive gelatine (1). 116 In this paper we describe the characterisation of a novel fining material which has the 117 potential to compete with isinglass in brewing applications. The novel finings is sourced 118 from hops, and can therefore be promoted as a natural ingredient of the brewing process; 119 although with conventional usage of hops in brewing the compounds believed to confer 120 fining activity would not typically persist into the product. Furthermore, the active material 121 is shown to be extractable from spent hops, the co-product generated through the 122 extraction of hop resins using liquid CO2 or ethanol. 6 123 2. Materials and methods 124 Chemicals 125 Analytical grade acetic acid, ferric ammonium sulfate, butanol, and High Performance Liquid 126 Chromatography (HPLC) grade acetone, acetonitrile, and ethyl acetate were purchased from 127 Fisher Scientific (Loughborough, UK).

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